Single-Cell Dissection of the SPP1-CD44 Axis Reveals Microglia-Astrocyte Crosstalk Driving Neuroinflammation in...

Temporal lobe epilepsy (TLE), one of the most prevalent focal epilepsies, is characterized by aberrant neuron and glial activation, yet the mechanisms driving microglia-astrocyte crosstalk remain elusive. To address this, we performed integrative single-nucleus RNA sequencing (snRNA-seq) analysis on surgically resected human brain tissue samples from a discovery cohort (4 TLE patients vs 4 controls) and a validation cohort (7 focal epilepsy cases vs the same controls). Using Seurat-based clustering, we identified 9 major cell types and further subclustered microglia and astrocytes. Cell-cell communication, gene regulatory networks, and pseudotime analysis were employed to explore the molecular mechanisms of microglia-astrocyte interactions. Results revealed significant expansion of both activated microglial and activated astrocytic subpopulations in TLE patients versus controls. The SPP1-CD44 axis emerged as the dominant pathway mediating their crosstalk, with reactive microglia as primary SPP1 senders and reactive astrocytes as CD44 receivers. The upstream regulators of SPP1-CD44 axis were subsequently explored, and 9 transcription factors (TFs) were identified as key regulators in reactive microglia. Pseudotime analysis further revealed a CD44-associated phenotypic shift from homeostatic to reactive astrocytes, characterized by progressive loss of synaptic regulatory functions and concurrent acquisition of neurotoxic properties during disease progression. Collectively, our multi-cohort snRNA-seq study reveals the SPP1-CD44 axis as a key mediator of neuroinflammatory pathology in TLE, linking microglial activation to astrocytic dysfunction. These findings broaden therapeutic strategies beyond neuronal targets, underscoring glial modulation as a promising adjunctive approach for epilepsy treatment.